The HIV-1 Tat protein is essential for viral replication and it acts to activate RNA polymerase II (RNAP II) elongation of the integrated provirus. To a considerable extent, Tat determines the amount of virus produced in the infected cell and this is an important feature of the pathogenesis of HIV-1 infection. Tat function is mediated by a cellular protein kinase termed P-TEFb that is composed of Cyclin T1 and Cdk9. Tat binds directly to Cyclin T1, and Tat/Cyclin T1/Cdk9 then associate with the viral TAR RNA structure that forms at the 5'ends of viral transcripts. When bound to TAR RNA, Cdk9 phosphorylates multiple substrates in the RNAP II complex, resulting in activation of elongation. Tat function is also mediated through its interaction with cellular factors that modify chromatin. Given its essential role in the viral life cycle and a mechanism of action that has features not found in cellular gene regulation, Tat remains an attractive but as yet unrealized target for anti-viral drugs. In the one-year funding period, we will continue our longstanding studies of Tat function. We will examine mechanisms involved in the establishment and maintenance of HIV-1 latent infection, as well as mechanisms involved in reaction of latent infection. We will determine if limiting levels of Cyclin T1 enhance the establishment of latent infection. We will screen the human kinome for kinases that are involved in reactivation of latent HIV-1 proviruses. Our initial results of this screen have identified candidate kinases that when over-expressed appear to reactivate a latent HIV-1 provirus. We will also investigate mechanisms that regulate Cdk9 T-loop phosphorylation in resting CD4+ T cells and therefore also regulate Tat function and HIV-1 replication. Completion of this research will elucidate molecular mechanisms involved in Tat function and HIV-1 replication. This will enhance our understanding of the HIV-1 life cycle and may serve as the foundation for novel therapeutic approaches to combat HIV/AIDS.